1. Field of the Invention
The invention relates generally to the field of immunology and immunotherapy applications using oligoribonucleotides as immune modulatory agents. More particularly, the invention relates to immune modulatory RNA compositions and methods of use thereof for modulating the immune response through Toll-like receptor 7 (TLR7).
2. Summary of the Related Art
The immune response involves both an innate and an adaptive response based upon the subset of cells involved in the response. For example, the T helper (Th) cells involved in classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs) are Th1 cells, whereas the Th cells involved as helper cells for B-cell activation are Th2 cells. The type of immune response is influenced by the cytokines and chemokines produced in response to antigen exposure. Cytokines provide a means for controlling the immune response by affecting the balance of T helper 1 (Th1) and T helper 2 (Th2) cells, which directly affects the type of immune response that occurs. If the balance is toward higher numbers of Th1 cells, then a cell-mediated immune response occurs, which includes activation of cytotoxic T cells (CTLs). When the balance is toward higher numbers of Th2 cells, then a humoral or antibody immune response occurs. Each of these immune response results in a different set of cytokines being secreted from Th1 and Th2 cells. Differences in the cytokines secreted by Th1 and Th2 cells may be the result of the different biological functions of these two T cell subsets.
Th1 cells are involved in the body's innate response to antigens (e.g., viral infections, intracellular pathogens, and tumor cells). The initial response to an antigen can be the secretion of IL-12 from antigen presenting cells (e.g., activated macrophages and dendritic cells) and the concomitant activation of Th1 cells. The result of activating Th1 cells is a secretion of certain cytokines (e.g., IL-2, IFN-gamma and other cytokines) and a concomitant activation of antigen-specific CTLs. Th2 cells are known to be activated in response to bacteria, parasites, antigens, and allergens and may mediate the body's adaptive immune response (e.g., immunoglobulin production and eosinophil activation) through the secretion of certain cytokines (e.g., IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 and other cytokines) and chemokines Secretion of certain of these cytokines may result in B-cell proliferation and an increase in antibody production. In addition, certain of these cytokines may stimulate or inhibit the release of other cytokines (e.g., IL-10 inhibits IFN-γ secretion from Th1 cells and IL-12 from dendritic cells). Ultimately, the balance between Th1 and Th2 cells and the cytokines and chemokines released in response to selected stimulus can have an important role in how the body's immune system responds to disease. For example, IFN-α may inhibit hepatitis C, and MIP-1α and MIP-1β (also known as CCL3 and CCL4 respectively) may inhibit HIV-1 infection. Optimal balancing of the Th1/Th2 immune response presents the opportunity to use the immune system to treat and prevent a variety of diseases.
The Th1 immune response can be induced in mammals for example by introduction of bacterial or synthetic DNA containing unmethylated CpG dinucleotides, which immune response results from presentation of specific oligonucleotide sequences (e.g., unmethylated CpG) to receptors on certain immune cells known as pattern recognition receptors (PRRs). Certain of these PRRs are Toll-like receptors (TLRs).
TLRs are intimately involved in inducing the innate immune response in response to microbial infection. In vertebrates, TLRs consist of a family of at least eleven proteins (TLR1 to TLR11) that are known to recognize pathogen associated molecular patterns (PAMP). Some TLRs are located on the cell surface to detect and initiate a response to extracellular pathogens and other TLRs are located inside the cell to detect and initiate a response to intracellular pathogens. Table 1 provides a representation of TLRs, the known agonists therefore and the cell types known to contain the TLR (Diebold, S. S. et al. (2004) Science 303:1529-31; Liew, F. et al. (2005) Nature 5:446-58; Hemmi, H. et al. (2002) Nat. Immunol. 3:196-200; Jurk, M. et al., (2002) Nat. Immunol. 3:499; Lee, J. et al. (2003) Proc. Natl. Acad. Sci. USA 100:6646-51; Alexopoulou, L. (2001) Nature 413:732-38).
TABLE 1Cell Types ContainingTLR MoleculeKnown AgonistReceptorCell SurfaceTLRs:TLR2bacterial lipopeptidesMonocytes/macrophagesMyeloid dendritic cellsMast cellsTLR4gram negative bacteriaMonocytes/macrophagesMyeloid dendritic cellsMast cellsIntestinal epitheliumTLR5motile bacteriaMonocyte/macrophagesDendritic cellsIntestinal epitheliumTLR6gram positive bacteriaMonocytes/macrophagesMast cellsB lymphocytesEndosomalTLRs:TLR3double stranded viral orDendritic cellscellular RNAB lymphocytesTLR7single stranded viral orMonocytes/macrophagescellular RNA;Plasmacytoid dendritic cellsRNA-immunoglobulinB lymphocytescomplexesTLR8single stranded viral orMonocytes/macrophagescellular RNA;Dendritic cellsRNA-immunoglobulinMast cellscomplexesTLR9DNA containingMonocytes/macrophagesunmethylatedPlasmacytoid dendritic cells“CpG” or synthetic motifs;B lymphocytesDNA-immunoglobulincomplexes
The signal transduction pathway mediated by the interaction between a ligand and a TLR is shared among most members of the TLR family and involves a toll/IL-1 receptor (TIR domain), the myeloid differentiation marker 88 (MyD88), IL-1R-associated kinase (IRAK), interferon regulating factor (IRF), TNF-receptor-associated factor (TRAF), TGFβ-activated kinase1, IκB kinases, IκB, and NF-κB (see, for example: Akira, S. (2003) J. Biol. Chem. 278:38105 and Geller et al. (2008) Curr. Drug Dev. Tech. 5:29-38). More specifically, for TLRs 1, 2, 4, 5, 6, 7, 8, 9, and 11, this signaling cascade begins with a PAMP ligand interacting with and activating the membrane-bound TLR, which exists as a homo-dimer in the endosomal membrane or the cell surface. Following activation, the receptor undergoes a conformational change to allow recruitment of the TIR domain containing protein MyD88, which is an adapter protein that is common to all TLR signaling pathways except TLR3. MyD88 recruits IRAK4, which phosphorylates and activates IRAK1. The activated IRAK1 binds with TRAF6, which catalyzes the addition of polyubiquitin onto TRAF6. The addition of ubiquitin activates the TAK/TAB complex, which in turn phosphorylates IRFs, resulting in NF-κB release and transport to the nucleus. NF-κB in the nucleus induces the expression of proinflammatory genes (see, for example, Trinchieri and Sher (2007) Nat. Rev. Immunol. 7:179-90).
The selective localization of TLRs and the signaling generated therefrom, provides some insight into their role in the immune response. The immune response involves both an innate and an adaptive response based upon the subset of cells involved in the response. For example, the T helper cells involved in classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs) are Th1 cells. This response is the body's innate response to antigen (e.g., viral infections, intracellular pathogens, and tumor cells), and results in a secretion of IFN-gamma and a concomitant activation of CTLs.
As a result of their involvement in regulating an inflammatory response, TLRs have been shown to play a role in the pathogenesis of many diseases, including autoimmunity, infectious disease, and inflammation (Papadimitraki et al. (2007) J. Autoimmun. 29:310-18; Sun et al. (2007) Inflam. Allergy Drug Targets 6:223-35; Diebold (2008) Adv. Drug Deliv. Rev. 60:813-23; Cook, D. N. et al. (2004) Nature Immunol. 5:975-79; Tse and Horner (2008) Semin. Immunopathol. 30:53-62; Tobias & Curtiss (2008) Semin. Immunopathol. 30:23-27; Ropert et al. (2008) Semin. Immunopathol. 30:41-51; Lee et al. (2008) Semin. Immunopathol. 30:3-9; Gao et al. (2008) Semin. Immunopathol. 30:29-40; Vijay-Kumar et al. (2008) Semin. Immunopathol. 30:11-21).
Studies have shown stimulation of an immune response using antisense oligonucleotides containing CpG dinucleotides (Zhao, Q. et al. (1996) Biochem. Pharmacol. 26:173-82). Subsequent studies showed that TLR9 recognizes unmethylated CpG motifs present in bacterial and synthetic DNA (Hemmi, H. et al. (2000) Nature 408:740-45). Other modifications of CpG-containing phosphorothioate oligonucleotides can also affect their ability to act through TLR9 and modulate the immune response (see, e.g., Zhao et al. (1996) Biochem. Pharmacol. 51:173-82; Zhao et al. (1996) Biochem. Pharmacol. 52:1537-44; Zhao et al. (1997) Antisense Nucleic Acid Drug Dev. 7:495-502; Zhao et al. (1999) Bioorg. Med. Chem. Lett. 9:3453-58; Zhao et al. (2000) Bioorg. Med. Chem. Lett. 10:1051-54; Yu et al. (2000) Bioorg. Med. Chem. Lett. 10:2585-88; Yu et al. (2001) Bioorg. Med. Chem. Lett. 11:2263-67; and Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-13). In addition, structure activity relationship studies have allowed identification of synthetic motifs and novel DNA-based structures that induce specific immune response profiles that are distinct from those resulting from unmethylated CpG dinucleotides. (Kandimalla, E. R. et al. (2005) Proc. Natl. Acad. Sci. USA 102:6925-30; Kandimalla, E. R. et al. (2003) Proc. Natl. Acad. Sci. USA 100:14303-08; Cong, Y. P. et al. (2003) Biochem. Biophys. Res. Commun. 310:1133-39; Kandimalla, E. R. et al. (2003) Biochem. Biophys. Res. Commun. 306:948-53; Kandimalla, E. R. et al. (2003) Nucleic Acids Res. 31:2393-400; Yu, D. et al. (2003) Bioorg. Med. Chem. 11:459-64; Bhagat, L. et al. (2003) Biochem. Biophys. Res. Commun. 300:853-61; Yu, D. et al. (2002) Nucleic Acids Res. 30:4460-69; Yu, D. et al. (2002) J. Med. Chem. 45:4540-48; Yu, D. et al. (2002) Biochem. Biophys. Res. Commun. 297:83-90; Kandimalla, E. R. et al. (2002) Bioconjug. Chem. 13:966-74; Yu, D. K. et al. (2002) Nucleic Acids Res. 30:1613-19; Yu, D. et al. (2001) Bioorg. Med. Chem. 9:2803-08; Yu, D. et al. (2001) Bioorg. Med. Chem. Lett. 11:2263-67; Kandimalla, E. R. et al. (2001) Bioorg. Med. Chem. 9:807-13; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett. 10:2585-88; Putta, M. R. et al. (2006) Nucleic Acids Res. 34:3231-38). However, until recently, natural ligands for TLR7 and TLR8 were unknown.
It has been shown that TLR7 and TLR8 recognize viral and synthetic single-stranded RNAs and small molecules, including a number of nucleosides (Diebold, S. S., et al. (2004) Science 303:1529-31). Diebold et al. show that the IFN-α response to influenza virus requires endosomal recognition of influenza genomic RNA and signaling by means of TLR7 and MyD88 and identify ssRNA as a ligand for TLR7. Certain synthetic compounds, the imidazoquinolones, imiquimod (R-837), and resiquimod (R-848) are ligands of TLR7 and TLR8 (Hemmi, H. et al. (2002) Nat. Immunol 3:196-200; Jurk, M. et al. (2002) Nat. Immunol 3:499). In addition, certain guanosine analogs, such as 7-deaza-G, 7-thia-8-oxo-G (TOG), and 7-allyl-8-oxo-G (loxoribine), have been shown to activate TLR7 at high concentrations (Lee, J. et al. (2003) Proc. Natl. Acad. Sci. USA 100:6646-51). However, these small molecules, e.g., imiquimod, are also known to act through other receptors (Schon, M. P. et al. (2006) J. Invest. Dermatol. 126:1338-47).
The lack of any known specific ssRNA motif for TLR7 or TLR8 recognition and the potentially wide range of stimulatory ssRNA molecules suggest that TLR7 and TLR8 can recognize both self and viral RNA. Recently it was shown that certain GU-rich oligoribonucleotides are immunostimulatory and act through TLR7 and TLR8 (Heil et al. (2004) Science 303:1526-29; Lipford et al., International Publication No. WO 03/086280; Wagner et al., International Publication No. WO 98/32462) when complexed with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N trimethylammoniummethylsulfate (DOTAP) or other lipid agents. However, RNA molecules have been used for many years, for example as ribozymes and, more recently, siRNA and microRNA, and RNA employed as ribozymes, siRNA, and microRNA contain GU dinucleotides. In addition, a number of these RNA molecules have been shown to elicit immune responses through TLR stimulation in the presence of lipids (Kariko et al. (2005) Immunity 23:165-75; Ma, Z. et al. (2005) Biochem. Biophys. Res. Commun. 330:755-59). However, the instability of these RNA molecules has hindered progress in using and applying these molecules in many areas (e.g., prevention and treatment of human disease).
Oligonucleotides and oligodeoxynucleotides containing a ribose or deoxyribose sugar have been used in a wide variety of fields, including but not limited to diagnostic probing, PCR priming, antisense inhibition of gene expression, siRNA, microRNA, aptamers, ribozymes, and immunotherapeutic agents based on Toll-like Receptors (TLRs). More recently, many publications have demonstrated the use of oligodeoxynucleotides as immune modulatory agents and their use alone or as adjuvants in immunotherapy applications for many diseases, such as allergy, asthma, autoimmunity, cancer, and infectious diseases.
The fact that DNA oligonucleotides are recognized by TLR9, while RNA oligonucleotides are recognized by TLR7 and/or TLR8 is most likely due to differences in the structural conformations between DNA and RNA. However, the chemical differences between DNA and RNA also make DNA far more chemically and enzymatically stable than RNA.
RNA is rapidly degraded by ubiquitous extracellular ribonucleases (RNases), which ensure that little, if any, self-ssRNA reaches the antigen-presenting cells. Exonuclease degradation of nucleic acids is predominantly of 3′-nuclease digestion with a smaller percentage through 5′-exonuclease action. In addition to exonuclease digestion, RNA can also be degraded by endonuclease activity of RNAses. RNA-based molecules have so far had to be complexed with lipids to provide stability against nucleases.
While providing an essential function of preventing autoimmune reactivity, these ribonucleases also present a substantial problem for any synthetic ssRNA molecule designed to be exploited for immunotherapy, as ribonucleases will rapidly degrade both synthetic and natural ssRNA. To overcome this hurdle, protection of ssRNA molecules from degradation has been attempted by encapsulating the ssRNA in liposomes, condensing it with polyethylenimine, or complexing it to molecules such as N-[1-(2,3 dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP). However, these protective measures are secondary measures applied to a still unstable ssRNA, and the effects of these protective measures on the in vivo efficacy and immune modulatory activity of ssRNA (natural or synthetic) remain unclear.
Agrawal et al. (U.S. Patent Application Publication No. 2008/0171712) describe a novel class of SIMRA compositions which bind to TLR7 and TLR8. However, a challenge remains to develop compounds that selectively bind to TLR7. Ideally, this challenge might be met through the design of inherently stable RNA-based molecules that can act as new immunotherapic agents, which will find use in a number of clinically relevant applications, such as improving the effects of vaccination when co-administered or treating and/or preventing diseases when invoking or enhancing an immune response is beneficial, for example cancer, autoimmune disorders, airway inflammation, inflammatory disorders, infectious diseases, skin disorders, allergy, asthma, or diseases caused by pathogens.